Cells require ordered movement of proteins and lipids from one membrane-bound compartment to another, while maintaining the organization, function and heterogeneity of the donor and acceptor membranes; vesicular membrane traffic carries out these functions. Clathrin coated vesicles are the most prominent form of traffic from the plasma membrane to endosomes (endocytosis), a pathway by which various ligands and their receptors enter cells. Clathrin coated vesicles are also important for traffic between endosomes and the trans-Golgi network (TGN). We propose to continue our studies of molecular mechanisms in clathrin-mediated membrane traffic, using direct observation by live-cell imaging with single-molecular sensitivity and high spatial and temporal resolution, to answer the following questions. (1) What are the molecular events required to initiate formation of a clathrin coated pit? (2) What are the molecular components of coats trapped at various stages of growth and closure: early and late abortive coated pits, and pits stalled at a relatively late stage, by imposing tension of the membrane and by inhibiting actin dynamics? (3) What is the mechanism of clathrin uncoating mediated by Hsc70 and its co-chaperone auxilin and how is the uncoating coordinated with assembly so that the two processes do not compete? PUBLIC HEALTH RELEVANCE: Vesicular membrane traffic is the principal mechanism for moving proteins and lipids among membrane-bound compartments in a cell. Clathrin coated vesicles are the most prominent form of traffic from the plasma membrane to endosomes (endocytosis), a pathway by which ligands such as hormones, transferrin, immunoglobulins, LDL, viruses and their receptors enter cells.